REGULATION OF APOAI GENE EXPRESSION BY NUCLEAR RECEPTORS

高密度脂蛋白(HDL)及其主要蛋白质组分， 载脂蛋白AI(ApoAI)在胆固醇动态平衡中起重要作用。 对调节载脂蛋白AI基因表达的分子机制的认识 在临床上很重要，因为降低血浆载脂蛋白AI和高密度脂蛋白水平 与过早的动脉粥样硬化和冠心病有关。我们的 长期目标是研究载脂蛋白AI的转录调控 基因并鉴定调节肝脏ApoAI合成的新信号 还有肠子。为了实现这一目标，我们发现监管要素 在ApoAI启动子与四个未知的核受体相互作用 配体、ARP-1、EAR-2、EAR-3和HNF-4以及形成的杂二聚体 在9-顺式维甲酸受体α(RXRpha)和ARP-1之间， EAR-2，EAR-3，全反式维甲酸受体α(RARpha)，维生素 D受体(VDR)，或过氧化物酶体增殖物激活受体(PPAR)。 ApoAI基因表达受ARP-1抑制，并被HNF-4和 然而，RXRpha，其他核受体对载脂蛋白AI的影响 表达方式是未知的。具体目标是：1)确定 结合载脂蛋白AI启动子的核受体的功能效应 该基因在肝脏和肠道细胞中的表达。2)绘制地图 HNF-4和ARP-1的功能结构域，并鉴定其基础 转录因子是这些核受体的靶标。3) 为了确定调节ARP-1 DNA结合活性的因素， EAR-2和EAR-3 实现这些目标的实验设计和方法如下：1) 表达上述核受体的重组质粒与 含ApoAI控制下的猫报告基因的构建 人肝癌(HepG2)和结肠癌(Caco-2)启动子区域的研究 2)细胞，采用磷酸钙共沉淀法。 RXRAlpha、RARAlpha、VDR和PPAR的传输将包括 9-顺式和全反式维甲酸、维生素的转基因后治疗 D；亚麻酸或亚油酸。2)缺失诱变 HNF-4和ARP-1的联合共转染实验将 用来绘制这些核的激活和抑制结构域 感受器。将使用蛋白质-蛋白质相互作用分析来研究 HNF-4和ARP-1与基础转录因子的相互作用 包括TATA结合蛋白(TBP)、TFIIB和其他与TBP相关的 因子(TAFs)，用于识别参与这些相互作用的域。 此外，交互克隆将被用于克隆组织特异性 可能与HNF-4或ARP-1相互作用的TAFs。3)生化检测和 将使用高效液相色谱来净化和 描述调节ARP-1的DNA结合活性的因素。 这些研究将加深我们对其分子机制的理解。 ApoAI通过核受体进行转录调控，并将揭示新的 调节载脂蛋白AI合成和贡献的信号转导通路 胆固醇动态平衡。此外，该信息可以提供 开发新的药理治疗策略的基础 监测常见的血脂紊乱。

High-density lipoprotein (HDL) and its major protein component, apolipoprotein AI (apoAI), play a major role in cholesterol homeostasis. Knowledge of the molecular mechanisms that regulate apoAI gene expression is clinically important because reduced plasma levels of apoAI and HDL are associated with premature atherosclerosis and coronary heart disease. Our long term objective is to study the transcriptional regulation of the apoAI gene and to identify novel signals that modulate apoAI synthesis in liver and intestine. Towards this goal, we have found that regulatory elements in the apoAI promoter interact with four nuclear receptors with unknown ligands, ARP-1, EAR-2, EAR-3, and HNF-4, and with heterodimers formed between the 9-cis retinoic acid receptor alpha (RXRalpha) and either ARP-1, EAR-2, EAR-3, all -trans retinoic acid receptor alpha (RARalpha), vitamin D receptor (VDR), or peroxisome proliferator-activated receptor (PPAR). ApoAI gene expression is repressed by ARP-1 and activated by HNF-4 and RXRalpha, however, the effects of the other nuclear receptors on apoAI expression are not known. The specific aims are: 1) To determine the functional effects of the nuclear receptors that bind to the apoAI promoter on the expression of this gene in hepatic and intestinal cells. 2) To map the functional domains of HNF-4 and ARP-1, and to identify the basal transcription factors which are targets for these nuclear receptors. 3) To identify the factors that regulate the DNA-binding activity of ARP-1, EAR-2, and EAR-3. The experimental design and methods for achieving these aims are; 1) Cotransfections of plasmids expressing the above nuclear receptors with constructs containing the CAT reporter gene under the control of the apoAI promoter region in human hepatoma (HepG2) and human colon carcinoma (Caco- 2) cells, using the calcium phosphate coprecipitation method. Transfections with RXRalpha, RARalpha, VDR, and PPAR will include posttransfection treatments with 9-cis and all-trans retinoic acid, vitamin D, and clofibric or linoleic acid, respectively. 2) Deletion mutagenesis of HNF-4 and ARP-1 in combination with cotransfection experiments will be employed to map the activation and repression domains of these nuclear receptors. Protein-protein interaction assays will be used to study the interactions of HNF-4 and ARP-1 with basal transcription factors., including the TATA-binding protein (TBP), TFIIB, and other TBP-associated factors (TAFs), an to identify the domains involved in these interactions. Furthermore, interaction cloning, will be used to clone tissue-specific TAFs that may interact with HNF-4 or ARP-1. 3) A biochemical assay and High performance liquid Chromatography will be employed to purify and characterize the factor that regulates the DNA-binding activity of ARP-1. These studies will enhance our understanding of the molecular mechanisms of apoAI transcriptional regulation by nuclear receptors and will reveal novel signal transduction pathways that modulate apoAI synthesis and contribute to cholesterol homeostasis. Furthermore, this information may provide a basis for the development of novel pharmacological strategies for monitoring common lipid disorders.